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研究生:戴書瑜
研究生(外文):Shu-Yu Dai
論文名稱:阿拉伯芥中一ANTHER DEHISCENCE REPRESSOR基因於過氧化體中抑制茉莉酸合成及次級細胞壁增厚以調控花藥開裂
論文名稱(外文):The Gene ANTHER DEHISCENCE REPRESSOR Controls Anther Dehiscence by Suppressing the Jasmonate Biosynthetic Pathway and Anther Cell Wall Thickening in the Peroxisomes of Arabidopsis
指導教授:楊長賢楊長賢引用關係
口試委員:林彩雲陳良築呂維茗李勇毅
口試日期:2017-07-19
學位類別:博士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:72
中文關鍵詞:花藥開裂過氧化體茉莉酸過氧化氫次級細胞壁
外文關鍵詞:anther dehiscenceperoxisomejasmonic acidH2O2sencondary wall
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植物中的雄不稔現象可由許多刺激造成,如荷爾蒙變化、逆境、基因突變等。於本人先前研究中發現阿拉伯芥中一雄不稔之基因Anther Dehiscence Repressor (ADR) 會受到荳蔻酸化修飾並可能參與調控花藥開裂,異位表現ADR於阿拉伯芥中造成花藥不開裂而導致雄不稔,而經由外加茉莉花酸於轉基因植株花苞可挽救該性狀。此外,調控花藥開裂之茉莉花酸其合成酵素的基因表現於轉殖株中亦明顯受到抑制,顯示ADR可能影響茉莉花酸之生合成。本研究則接續ADR進一步的功能性分析。35S:ADR+GFP轉殖菸草葉片中觀察到ADR分布於過氧化體,無法受到荳蔻酸化修飾的35S::ADR-Gly+GFP則停留於胞質中,且35S::ADR-Gly轉殖株並無花藥未開裂之性狀。35S::ADR轉殖株花藥的木質素染色顯示花藥內皮層之次級細胞壁並無增厚,而活性氧物質 (reactive oxygen species, ROS) 中過氧化氫 (H2O2) 的累積為次級細胞壁增厚所需之前置步驟,此過程亦受到影響,此外調控次級細胞壁增厚的NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1) 及NST2其表現亦受到抑制。綜合上述結果我們認為ADR在受到荳蔻酸化修飾後會與過氧化體的膜結合,並藉由抑制茉莉花酸活性、H2O2之累積與NST1/NST2表現來調控花藥開裂。
Male-sterility in plants is caused by various stimuli such as hormone change, stress, cytoplasmic and nuclear mutations. Our previous study showed a gene Anther Dehiscence Repressor (ADR) which is modified by N-myristoylation may be involved in regulating male-sterility in Arabidopsis. 35S::ADR transgenic Arabidopsis showed male-sterility due to anther indehiscence. The male-sterility of 35S::ADR was rescued by the application of JA to the transgenic floral buds. Besides, genes participate in JA (jasmonic acid) biosynthesis were suppressed in 35S::ADR transgenic Arabidopsis, indicating that ADR may affect the biosynthesis of JA. In this study, functional analysis of ADR was continued. 35S::ADR+GFP fusion proteins, which can be modified by N-myristoylation, were able to target to peroxisomes, whereas the 35S::ADR-Gly+GFP mutant proteins which lack the N-terminal Gly accumulated in the cytosol and were absent in the peroxisomes. 35S::ADR-Gly that failed to be myristoylated showed normal fertility as that observed in wild-type plants. Lignin staining of anthers showed no secondary thickening or lignification in the anther endothecium of 35S::ADR plant compared to wild-type. 35S::ADR also reduced the ROS accumulation and suppressed the expression of NST1 and NST2 that is necessary for anther dehiscence by regulating the secondary wall thickening in endothetical cells of anthers. These results demonstrated that ADR needs to be N-myristoylated and targeted to the peroxisome to negatively regulate anther dehiscence by suppressing JA activity, ROS accumulation and NST1/NST2 expression.
中文摘要-------------------------------------------------i
Abstract-------------------------------------------ii
目次 ------------------------------------ iii
前言--------------------------------------------------------------------------------------------------1
材料與方法-----------------------------------------------------------------------------------------5
一、實驗材料------------------------------------------------------------------------------------5
二、阿拉伯芥的種植---------------------------------------------------------------------------5
三、菸草的種植-----------------------------------------------------------------------------5
四、掃描式電子顯微鏡觀察--------------------------------------------------------------5
五、阿拉伯芥中基因的選殖-----------------------------------------------------------------6
(一) 阿拉伯芥總體核糖核酸 (total RNA) 之萃取-------------------------------------6
(二) 反轉錄增幅反應 (Reverse Transcription,RT) --------------------------------------6
(三) 聚合酶鏈鎖反應 (Polymerase Chain Reaction, PCR) ----------------------------6
(四) DNA片段純化與回收------------------------------------------------------------------7
(五) 接合反應 (Ligation) --------------------------------------------------------------------7
(六) 勝任細胞之製備 (Competent cell preparation) ------------------------------------7
(七) 細胞轉型作用 (transformation) ------------------------------------------------------8
(八) 轉型細菌菌落進行聚合酶鏈鎖反應篩選 (colony PCR) ------------------------8
(九) 高純度質體DNA的抽取--------------------------------------------------------------8
(十) 酵素截切 (digestion) ----------------------------------------------------------------9
(十一) DNA定序 (autosequencing) -------------------------------------------------------9
六、阿拉伯芥基因轉殖與篩選轉基因植物-----------------------------------------------9
(一) 農桿菌勝任細胞之配製--------------------------------------------------------------9
(二) 農桿菌快速冷凍轉型-----------------------------------------------------------------9
(三) 阿拉伯芥之轉殖-----------------------------------------------------------------------10
(四) 快速大量篩選轉殖植株--------------------------------------------------------------10
七、木質素染色------------------------------------------------------------------------------10
八、轉基因植株中相關基因的表現測定-------------------------------------------------10
(一) 反轉錄定量 (RT-PCR) -----------------------------------------------------------------10
(二) 即時定量分析 (Real-time PCR) ------------------------------------------------------10
九、3,3’-Diaminobenzidine tetrahydrochloride hydrate (DAB) 染色-------------------11
十、共軛焦顯微鏡 (Confocal) ------------------------------------------------------------11
十一、菸草基因轉殖實驗之農桿菌注射 ---------------------------11
十二、H2O2處理-------------------------------------------------------------------------------12
十三、茉莉酸定量 (JA quantification) -----------------------------------------------------13
結果-----------------------------------------------------------------------------------------------12
一、ADR相似基因ARG (ADR-REDUNDANT GENE) 之功能性分析------------13
二、ADR與ARG共同突變株 (adr/arg-15) 之構築與分析----------------14
三、35S::ADR基因轉殖植株其花藥與種子之掃描式電子顯微鏡觀察------------14
四、35S::ADR基因轉殖植株與野生型阿拉伯芥之雜交 (cross) 實驗--------------15
五、野生型阿拉伯芥中ADR於花藥各時期之表現測定-----------------------------15
六、ADR與CAT3於菸草之暫時性表達-------------------------------------15
七、ADR+GUS+GFP與ADR-Gly+GUS+GFP於菸草之暫時性表達--------------16
八、35S::ADR-VP16與35S::ADR-SRDX之構築與植物基因轉殖-------------------17
九、ADR結合至過氧化體與花藥開裂性狀之關連性分析---------------------------18
十、35S::ADR植株中H2O2累積情形之分析------------------------------------------18
十一、35S::ADR植株H2O2之處理--------------------------------------------------------19
十二、35S::ADR植株中木質素堆積與次級細胞壁增厚之觀察----------------------19
十三、35S::ADR植株中NST1及NST2表現量分析------------------------------------20
十四、35S::ADR植株中OPR3+GFP之表現位置分析-------------------------------20
討論-------------------------------------------------------------------------------------------------22
參考文獻-------------------------------------------------------------------------------------------25
表格-------------------------------------------------------------------------------------------------33
表1. 本研究中使用之引子序列------------------------------------------------------------33
圖表-------------------------------------------------------------------------------------------------34
圖1. ADR與At3g23930胺基酸序列比對--------------------------------------------------34
圖2. ARG之分子選殖膠體電泳圖----------------------------------------------------------35
圖3. ARG之T-DNA插入位置與突變株鑑定之膠體電永圖---------------------------36
圖4. ARG基因於35S::ARG與ARG之T-DNA突變株中表現量分析---------------37
圖5. ADR及ARG於adr/arg-15雙突變株中表現量分析-------------------------------38
圖6. 35S::ADR植株與野生型阿拉伯芥之花粉外觀-------------------------------------39
圖7. 35S::ADR植株與野生型阿拉伯芥進行人工授粉之結果-------------------------40
圖8. ADR於野生型阿拉伯芥花藥不同時期之表現量分析-----------------------------41
圖9. CAT3之分子選殖膠體電泳圖---------------------------------------------------------42
圖10. 35S::CAT3+mORG2與35S::ADR+GFP轉殖菸草葉片之螢光表現位置-----43
圖11. ADR+GUS+GFP及ADR-Gly+GUS+GFP之分子選殖膠體電泳圖------------44
圖12. 35S::ADR+GUS+GFP及35S::ADR-Gly+GUS+GFP菸草葉片螢光表現-----45
圖13. ADR-SRDX與ADR-VP16之分子選殖膠體電泳圖-------------------------------46
圖14. 35S::ADR-Gly與35S::ADR之基因轉殖植株性狀分析--------------------------47
圖15. 35S::ADR植株與野生型阿拉伯芥花序處理H2O2後之DAB染色-----------48
圖16. 35S::ADR植株與野生型阿拉伯芥之DAB染色----------------------------------49
圖17. 35S::ADR植株之H2O2染色----------------------------------------------------------50
圖18. 35S::ADR植株與野生型阿拉伯芥花藥之木質素染色---------------------------51
圖19. 35S::ADR植株與野生型阿拉伯芥花朵中NST1與NST2表現量分析--------52
圖20. OPR3-12H之分子選殖膠體電泳圖-------------------------------------------------53
圖21. ADR之胺基酸序列---------------------------------------------------------------------54
圖22. ADR調控花藥開裂之功能模式圖--------------------------------------------------55
附圖-------------------------------------------------------------------------------------------------56
附圖1. 花藥構造示意圖----------------------------------------------------------------------56
附圖2. 花藥開裂過程-------------------------------------------------------------------------57
附圖3. 茉莉酸生合成路徑-------------------------------------------------------------------58
附圖4. 35S::ADR基因轉殖植株因花藥不開裂造成之雄不稔性狀-------------------59
附圖5. pADR-GUS在野生型阿拉伯芥花器之GUS染色分析-------------------------60
附圖6. 35S::ADR+GFP與35S::ADR-Gly+GFP轉殖菸草葉片之GFP表現--------61
附圖7. 參與茉莉花酸生合成途徑中各基因在35S::ADR花苞之表現---------------62
附圖8. 35S::ADR的不孕轉殖株之茉莉花酸處理分析----------------------------------63
附圖9. pGEM®-T Easy vector圖譜----------------------------------------------------------64
附圖10. pEpyon-12K之圖譜-----------------------------------------------------------------65
附圖11. 35S::ADR基因轉殖植株與野生型植株之花粉活性測定染色---------------66
附圖12. pEpyon-33K之圖譜-----------------------------------------------------------------67
附圖13. pEpyon-32H之圖譜-----------------------------------------------------------------68
附圖14. pEpyon-32K之圖譜-----------------------------------------------------------------69
附圖15. pEpyon-2aK之圖譜-----------------------------------------------------------------70
附圖16. pEpyon-2bK之圖譜-----------------------------------------------------------------71
附圖17. pEpyon-12H之圖譜-----------------------------------------------------------------72
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